Virtual screening–based discovery of AI-2 quorum sensing inhibitors that interact with an allosteric hydrophobic site of LsrK and their functional evaluation
Introduction: Quorum sensing (QS) is a bacterial intracellular and intercellular communication system that regulates virulence factor production, biofilm formation, and antibiotic sensitivity. Quorum-sensing inhibitors (QSIs) are a novel class of antibiotics that can effectively combat antibiotic re...
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| Format: | Article |
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Frontiers Media S.A.
2023-05-01
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| Series: | Frontiers in Chemistry |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fchem.2023.1185224/full |
| _version_ | 1797821499701198848 |
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| author | Qianqian Shi Qianqian Shi Huiqi Wen Yijie Xu Yijie Xu Xu Zhao Jing Zhang Ye Li Qingbin Meng Fang Yu Fang Yu Junhai Xiao Junhai Xiao Xingzhou Li Xingzhou Li |
| author_facet | Qianqian Shi Qianqian Shi Huiqi Wen Yijie Xu Yijie Xu Xu Zhao Jing Zhang Ye Li Qingbin Meng Fang Yu Fang Yu Junhai Xiao Junhai Xiao Xingzhou Li Xingzhou Li |
| author_sort | Qianqian Shi |
| collection | DOAJ |
| description | Introduction: Quorum sensing (QS) is a bacterial intracellular and intercellular communication system that regulates virulence factor production, biofilm formation, and antibiotic sensitivity. Quorum-sensing inhibitors (QSIs) are a novel class of antibiotics that can effectively combat antibiotic resistance. Autoinducer-2 (AI-2) is a universal signaling molecule that mediates inter- and intraspecies QS systems among different bacteria. Furthermore, LsrK plays an important role in regulating the activity and stability of the intracellular AI-2 signaling pathway. Thus, LsrK is considered an important target for the development of QSIs.Methods: We designed a workflow integrating molecular dynamic (MD) simulations, virtual screening, LsrK inhibition assays, cell-based AI-2-mediated QS interference assays, and surface plasmon resonance (SPR)-based protein affinity assays to screen for potential LsrK kinase inhibitors.Results: MD simulation results of the LsrK/ATP complex revealed hydrogen bonds and salt bridge formation among four key residues, namely, Lys 431, Tyr 341, Arg 319, and Arg 322, which are critical for the binding of ATP to LsrK. Furthermore, MD simulation results indicated that the ATP-binding site has an allosteric pocket that can become larger and be occupied by small molecule compounds. Based on these MD simulation results, a constraint of forming at least one hydrogen bond with Arg 319, Arg 322, Lys 431, or Tyr 341 residues was introduced when performing virtual screening using Glide’s virtual screening workflow (VSW). In the meantime, compounds with hydrophobic group likely to interact with the allosteric hydrophobic pocket are preferred when performing visual inspection. Seventy-four compounds were selected for the wet laboratory assays based on virtual screening and the absorption, distribution, metabolism, and excretion (ADME) properties of these compounds. LsrK inhibition assays revealed 12 compounds inhibiting LsrK by more than 60% at a 200 μM concentration; four of these (Y205-6768, D135-0149, 3284–1358, and N025-0038) had IC50 values below 50 μM and were confirmed as ATP-competitive inhibitors. Six of these 12 LsrK inhibitors exhibited high AI-2 QS inhibition, of which, Y205-6768 had the highest activity with IC50 = 11.28 ± 0.70 μM. The SPR assay verified that compounds Y205-6768 and N025-0038 specifically bound to LsrK. MD simulation analysis of the docking complexes of the four active compounds with LsrK further confirmed the importance of forming hydrogen bonds and salt bridges with key basic amino acid residues including Lys 431, Tyr 341, Arg 319, and Arg 322 and filling the allosteric hydrophobic pocket next to the purine-binding site of LsrK.Discussion: Our study clarified for the first time that there is an allosteric site near the ATP-binding site of Lsrk and that it enriches the structure–activity relationship information of Lsrk inhibitors. The four identified compounds showed novel structures, low molecular weights, high activities, and novel LsrK binding modes, rendering them suitable for further optimization for effective AI-2 QSIs. Our work provides a valuable reference for the discovery of QSIs that do not inhibit bacterial growth, thereby avoiding the emergence of drug resistance. |
| first_indexed | 2024-03-13T09:53:35Z |
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| institution | Directory Open Access Journal |
| issn | 2296-2646 |
| language | English |
| last_indexed | 2024-03-13T09:53:35Z |
| publishDate | 2023-05-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Chemistry |
| spelling | doaj.art-6d67397a389348c1bfb949a1601db0332023-05-24T05:16:17ZengFrontiers Media S.A.Frontiers in Chemistry2296-26462023-05-011110.3389/fchem.2023.11852241185224Virtual screening–based discovery of AI-2 quorum sensing inhibitors that interact with an allosteric hydrophobic site of LsrK and their functional evaluationQianqian Shi0Qianqian Shi1Huiqi Wen2Yijie Xu3Yijie Xu4Xu Zhao5Jing Zhang6Ye Li7Qingbin Meng8Fang Yu9Fang Yu10Junhai Xiao11Junhai Xiao12Xingzhou Li13Xingzhou Li14School of Petrochemical Engineering, Liaoning Petrochemical University, Fushun, ChinaNational Engineering Research Center for the Emergency Strategic Drug, Beijing Institute of Pharmacology and Toxicology, Beijing, ChinaState Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing, ChinaNational Engineering Research Center for the Emergency Strategic Drug, Beijing Institute of Pharmacology and Toxicology, Beijing, ChinaState Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology and Toxicology, Beijing, ChinaDepartment of Hepatology, Fifth Medical Center of Chinese PLA General Hospital, Beijing, ChinaQionglai Medical Center Hospital, Chengdu, ChinaThe No 968 Hospital of PLA, Jinzhou, ChinaState Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology and Toxicology, Beijing, ChinaSchool of Petrochemical Engineering, Liaoning Petrochemical University, Fushun, ChinaNational Engineering Research Center for the Emergency Strategic Drug, Beijing Institute of Pharmacology and Toxicology, Beijing, ChinaNational Engineering Research Center for the Emergency Strategic Drug, Beijing Institute of Pharmacology and Toxicology, Beijing, ChinaState Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology and Toxicology, Beijing, ChinaNational Engineering Research Center for the Emergency Strategic Drug, Beijing Institute of Pharmacology and Toxicology, Beijing, ChinaState Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology and Toxicology, Beijing, ChinaIntroduction: Quorum sensing (QS) is a bacterial intracellular and intercellular communication system that regulates virulence factor production, biofilm formation, and antibiotic sensitivity. Quorum-sensing inhibitors (QSIs) are a novel class of antibiotics that can effectively combat antibiotic resistance. Autoinducer-2 (AI-2) is a universal signaling molecule that mediates inter- and intraspecies QS systems among different bacteria. Furthermore, LsrK plays an important role in regulating the activity and stability of the intracellular AI-2 signaling pathway. Thus, LsrK is considered an important target for the development of QSIs.Methods: We designed a workflow integrating molecular dynamic (MD) simulations, virtual screening, LsrK inhibition assays, cell-based AI-2-mediated QS interference assays, and surface plasmon resonance (SPR)-based protein affinity assays to screen for potential LsrK kinase inhibitors.Results: MD simulation results of the LsrK/ATP complex revealed hydrogen bonds and salt bridge formation among four key residues, namely, Lys 431, Tyr 341, Arg 319, and Arg 322, which are critical for the binding of ATP to LsrK. Furthermore, MD simulation results indicated that the ATP-binding site has an allosteric pocket that can become larger and be occupied by small molecule compounds. Based on these MD simulation results, a constraint of forming at least one hydrogen bond with Arg 319, Arg 322, Lys 431, or Tyr 341 residues was introduced when performing virtual screening using Glide’s virtual screening workflow (VSW). In the meantime, compounds with hydrophobic group likely to interact with the allosteric hydrophobic pocket are preferred when performing visual inspection. Seventy-four compounds were selected for the wet laboratory assays based on virtual screening and the absorption, distribution, metabolism, and excretion (ADME) properties of these compounds. LsrK inhibition assays revealed 12 compounds inhibiting LsrK by more than 60% at a 200 μM concentration; four of these (Y205-6768, D135-0149, 3284–1358, and N025-0038) had IC50 values below 50 μM and were confirmed as ATP-competitive inhibitors. Six of these 12 LsrK inhibitors exhibited high AI-2 QS inhibition, of which, Y205-6768 had the highest activity with IC50 = 11.28 ± 0.70 μM. The SPR assay verified that compounds Y205-6768 and N025-0038 specifically bound to LsrK. MD simulation analysis of the docking complexes of the four active compounds with LsrK further confirmed the importance of forming hydrogen bonds and salt bridges with key basic amino acid residues including Lys 431, Tyr 341, Arg 319, and Arg 322 and filling the allosteric hydrophobic pocket next to the purine-binding site of LsrK.Discussion: Our study clarified for the first time that there is an allosteric site near the ATP-binding site of Lsrk and that it enriches the structure–activity relationship information of Lsrk inhibitors. The four identified compounds showed novel structures, low molecular weights, high activities, and novel LsrK binding modes, rendering them suitable for further optimization for effective AI-2 QSIs. Our work provides a valuable reference for the discovery of QSIs that do not inhibit bacterial growth, thereby avoiding the emergence of drug resistance.https://www.frontiersin.org/articles/10.3389/fchem.2023.1185224/fullATP competitive inhibitorsLsrKantibacterial agentsquorum sensingvirtual screeningmolecular dynamics |
| spellingShingle | Qianqian Shi Qianqian Shi Huiqi Wen Yijie Xu Yijie Xu Xu Zhao Jing Zhang Ye Li Qingbin Meng Fang Yu Fang Yu Junhai Xiao Junhai Xiao Xingzhou Li Xingzhou Li Virtual screening–based discovery of AI-2 quorum sensing inhibitors that interact with an allosteric hydrophobic site of LsrK and their functional evaluation Frontiers in Chemistry ATP competitive inhibitors LsrK antibacterial agents quorum sensing virtual screening molecular dynamics |
| title | Virtual screening–based discovery of AI-2 quorum sensing inhibitors that interact with an allosteric hydrophobic site of LsrK and their functional evaluation |
| title_full | Virtual screening–based discovery of AI-2 quorum sensing inhibitors that interact with an allosteric hydrophobic site of LsrK and their functional evaluation |
| title_fullStr | Virtual screening–based discovery of AI-2 quorum sensing inhibitors that interact with an allosteric hydrophobic site of LsrK and their functional evaluation |
| title_full_unstemmed | Virtual screening–based discovery of AI-2 quorum sensing inhibitors that interact with an allosteric hydrophobic site of LsrK and their functional evaluation |
| title_short | Virtual screening–based discovery of AI-2 quorum sensing inhibitors that interact with an allosteric hydrophobic site of LsrK and their functional evaluation |
| title_sort | virtual screening based discovery of ai 2 quorum sensing inhibitors that interact with an allosteric hydrophobic site of lsrk and their functional evaluation |
| topic | ATP competitive inhibitors LsrK antibacterial agents quorum sensing virtual screening molecular dynamics |
| url | https://www.frontiersin.org/articles/10.3389/fchem.2023.1185224/full |
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